|
| Status |
Public on May 04, 2017 |
| Title |
iPTL myometrial epigenome |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
5mC MeDIP myometrium of women with idiopathic preterm laboring (iPTL); London
|
| Organism |
Homo sapiens |
| Characteristics |
pregnancy: idiopathic preterm laboring (iPTL)
tissue: myometrium
term length: preterm
with or without labor: laboring
antibody: 5-methylcytosine (5mC) mAb (Eurogentec)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Myometrial tissue was collected from the upper margin of a lower segment uterine incision at cesarean section as we have previously described. Any decidua and serosa were removed, and the remaining myometrium was flash frozen in liquid nitrogen and stored at -80°C before analysis. Labor was defined as the presence of regular uterine contractions (every 3–4 min), resulting in cervical effacement and dilation. The myometrial epigenome was compared in six groups of women: term laboring (TL, n = 11); term no labor consisting of two sets of tissues, one collected in San Antonio (TNL-san, n = 10) and one collected in London (TNL-lon, n = 9); preterm no labor (PTNL, n = 10); idiopathic preterm laboring (iPTL, n = 6); and twin gestations with preterm laboring (tPTL, n = 7). Genomic DNA was isolated from each myometrial tissue sample using DNeasy spin columns (Qiagen). Following isolation, DNA quality and quantity of all the isolations were measured using NanoDrop 2000 (Thermo Fisher Scientific) and each DNA sample was routinely examined by agarose gel electrophoresis with ethidium bromide staining to verify the absence of contaminating RNA and to determine the extent of genomic DNA degradation. Genomic DNA samples in each of the six groups were combined, resulting in six pools of DNA that were then subjected to methylated DNA immunoprecipitation (MeDIP) analysis.
|
| Label |
Cy5
|
| Label protocol |
Fragmented genomic DNA (10 ug) was denatured and immunoprecipitated using the mouse monoclonal antibody against 5-methylcytosine (clone 33D3, Eurogentec). A portion of the fragmented DNA (20%) was left untreated to serve as input control. Immunoprecipitated and input DNA were amplified using GenomePlex Complete Whole Genome Amplification (WGA2) Kit (Sigma-Aldrich). After cleanup with QIAquick PCR Purification columns (Qiagen), immunoprecipitated and input DNA were denatured and differentially labeled with fluorescent Cy5 and Cy3 dyes, respectively, using Dual-Color DNA Labeling Kit (Roche NimbleGen).
|
| |
|
| Channel 2 |
| Source name |
Input myometrium of women with idiopathic preterm laboring (iPTL); London
|
| Organism |
Homo sapiens |
| Characteristics |
pregnancy: idiopathic preterm laboring (iPTL)
tissue: myometrium
term length: preterm
with or without labor: laboring
antibody: none
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Myometrial tissue was collected from the upper margin of a lower segment uterine incision at cesarean section as we have previously described. Any decidua and serosa were removed, and the remaining myometrium was flash frozen in liquid nitrogen and stored at -80°C before analysis. Labor was defined as the presence of regular uterine contractions (every 3–4 min), resulting in cervical effacement and dilation. The myometrial epigenome was compared in six groups of women: term laboring (TL, n = 11); term no labor consisting of two sets of tissues, one collected in San Antonio (TNL-san, n = 10) and one collected in London (TNL-lon, n = 9); preterm no labor (PTNL, n = 10); idiopathic preterm laboring (iPTL, n = 6); and twin gestations with preterm laboring (tPTL, n = 7). Genomic DNA was isolated from each myometrial tissue sample using DNeasy spin columns (Qiagen). Following isolation, DNA quality and quantity of all the isolations were measured using NanoDrop 2000 (Thermo Fisher Scientific) and each DNA sample was routinely examined by agarose gel electrophoresis with ethidium bromide staining to verify the absence of contaminating RNA and to determine the extent of genomic DNA degradation. Genomic DNA samples in each of the six groups were combined, resulting in six pools of DNA that were then subjected to methylated DNA immunoprecipitation (MeDIP) analysis.
|
| Label |
Cy3
|
| Label protocol |
Fragmented genomic DNA (10 ug) was denatured and immunoprecipitated using the mouse monoclonal antibody against 5-methylcytosine (clone 33D3, Eurogentec). A portion of the fragmented DNA (20%) was left untreated to serve as input control. Immunoprecipitated and input DNA were amplified using GenomePlex Complete Whole Genome Amplification (WGA2) Kit (Sigma-Aldrich). After cleanup with QIAquick PCR Purification columns (Qiagen), immunoprecipitated and input DNA were denatured and differentially labeled with fluorescent Cy5 and Cy3 dyes, respectively, using Dual-Color DNA Labeling Kit (Roche NimbleGen).
|
| |
|
| |
| Hybridization protocol |
Equal amounts (34 ug) of Cy5- and Cy3-labeled samples were combined and hybridized to each Human DNA Methylation 2.1M v2 genome tilling array (Roche NimbleGen) for 16-20 h at 42˚C using NimbleGen hybridization System 4. After washing, the hybridized arrays were dried using ArrayIt high-speed microarray centrifuge.
|
| Scan protocol |
Arrays were scanned on NimbleGen MS 200 microarray scanner using NimbleGen Data Collection software.
|
| Description |
5mC MeDIP myometrium of women with idiopathic preterm laboring (iPTL); London
|
| Data processing |
Arrays were processed using the standard protocol (NimbleGen Arrays User’s Guide for DNA methylation analysis v1.0.
log2 (MeDIP/input) ratio in gff files.
|
| |
|
| Submission date |
Jul 20, 2015 |
| Last update date |
May 04, 2017 |
| Contact name |
Kohzoh Mitsuya |
| E-mail(s) |
mitsuya@uthscsa.edu
|
| Phone |
210-567-7064
|
| Organization name |
University of Texas Health Science Center San Antonio
|
| Department |
Center for Pregnancy and Newborn Research
|
| Lab |
Leslie Myatt
|
| Street address |
7703 Floyd Curl Drive
|
| City |
San Antonio |
| State/province |
Texas |
| ZIP/Postal code |
78229-3900 |
| Country |
USA |
| |
|
| Platform ID |
GPL16284 |
| Series (1) |
| GSE71123 |
MeDIP assay using human myometrium of women with preterm labor of different phenotypes vs. normal term labor |
|
